Problem: Lower gastrointestinal bleeding is a significant medical problem in the United States, accounting for more than 120,000 hospital admissions and tens of billions of dollars of healthcare spending annually. It is common for bleeding from a single site to stop and restart multiple times. The three modalities now used to diagnose and locate gastrointestinal bleeding fail 50% of the time, largely because they all require a minimum rate of bleeding at the time of the test. Without locating the source, the patient continues to be at risk for further bleeding. Approach: Our initial proof-of-principle in ivo studies illustrate that a radiolabeled fibrinogen- based molecular imaging tracer will concentrate at the injured blood vessel wall as part of the adherent clot, and the tracer can be used to locate bleeding sites even after bleeding has stopped. To advance the fibrinogen-based tracer concept towards a clinical test, we propose to leverage single photon emission computed tomography and develop a tracer consisting of fibrinogen labeled with technetium-99m. We propose to develop and test a high-efficiency protocol to produce biologically active [99mTc]-fibrinogen. An iterative set of experiments would maximize yield and specific activity of the radiolabeling process while maintaining stability and biological activity of the tracer. Using the optimized process to produce an injectable SPECT tracer, we would next demonstrate the efficacy of [99mTc]-fibrinogen to detect bleeding in an animal model. Bleeding at a biopsy site in the descending colon of a rat will be located using SPECT imaging; the results will be confirmed by radiometric measurements of excised tissues. Impact: The proposed technology represents a shift in the clinical paradigm for detecting lower gastrointestinal bleeding because it locates injured blood vessels, not bleeding itself. Our ultimate clinical product would consist of a radiopharmaceutical preparatory kit for preparing a single patient dose of the SPECT tracer using a 99mTc generator. The tracer will be administered intravenously and localized using a nuclear medicine camera. The 99mTc generator and camera are standard equipment in most hospitals. Primary use will be for accurate diagnosis of lower gastrointestinal bleeding and possibly bleeding due to other disorders. A rapid and accurate test to locate bleeding sites would transform the standard care, measured as lower cost of healthcare due to fewer test procedures and associated complications, shorter in-patient days, and reduced morbidity and mortality in patient outcomes.